RESEARCH Open Access

Identification of two new protective pre-erythrocytic malaria vaccine antigen candidatesKeith Limbach1,2*, Joao Aguiar1,2, Kalpana Gowda1, Noelle Patterson1,2, Esteban Abot1,2, Martha Sedegah1,John Sacci3 and Thomas Richie1

Abstract

Background: Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing thedevelopment of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens.To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response againstmultiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of themalaria vaccine antigens that have been evaluated in people have not elicited a protective immune response,there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stagemalaria antigens was evaluated in a Plasmodium yoelii/mouse protection model.

Methods: Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all threeP. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated forsubsequent infection.

Results: Vaccines that expressed any one of the three antigens did not protect a high percentage of mice againsta P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of micethan a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigenvaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protectionelicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the singleantigen vaccines, suggesting a potential synergistic interaction.

Conclusions: This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.

BackgroundMalaria kills approximately 863,000 people every year [1].Although a variety of anti-malarial drugs exist, the costof these drugs can be prohibitive in the relatively poorareas of the world where malaria is endemic. The wide-spread use of the most commonly employed drugs hasalso resulted in the expansion of drug-resistant parasites,rendering many of these drugs ineffective [2]. In theabsence of inexpensive, highly potent drugs, vaccinationrepresents the most cost-effective way of supplementingtraditional malaria interventions.A successful malaria vaccine will need to protect people

against a large population of antigenically diverse malaria

parasites. A vaccine based on a single isolate of a singleantigen may not be able to elicit an immune response thatis broad enough to protect individuals against this hetero-geneous population. Therefore, an efficacious malariavaccine may need to induce an immune response againstmultiple malaria antigens, a belief that has propelled thedevelopment of whole-organism malaria vaccines, such asthe irradiated sporozoite vaccine and the genetically atte-nuated sporozoite vaccine [3,4].A variety of malaria vaccine candidates are being eval-

uated in clinical trials throughout the world. The mostadvanced vaccine candidate, RTS,S, is currently beingevaluated in a phase 3 trial at 11 sites in seven Africancountries. RTS,S is a recombinant protein vaccine basedon the Plasmodium falciparum circumsporozoite pro-tein (CSP). It has protected malaria-naïve adults againstan experimental P. falciparum challenge and reduced

* Correspondence: keith.limbach@med.navy.mil1U.S. Military Malaria Vaccine Program, Naval Medical Research Center, 503Robert Grant Avenue, Silver Spring, MD, USAFull list of author information is available at the end of the article

Limbach et al. Malaria Journal 2011, 10:65http://www.malariajournal.com/content/10/1/65

© 2011 Limbach et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

mailto:keith.limbach@med.navy.milhttp://creativecommons.org/licenses/by/2.0

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malaria-associated episodes in children living in malariaendemic areas [5,6]. The level and duration of immunityinduced by RTS,S, however, is relatively modest.One way to potentially enhance the efficacy of RTS,S,

or any other subunit malaria vaccine, would be to incor-porate additional malaria antigens into the vaccine,thereby broadening the immune response elicited by thevaccine. At least one other malaria antigen has protectedvolunteers against a malaria challenge. A prime-boostregimen with adenovirus and poxvirus vectors expres-sing P. falciparum thrombospondin-related adhesiveprotein (TRAP) has protected volunteers against anexperimental P. falciparum challenge [7]. A prime-boostregimen with DNA and adenovirus vectors expressingCSP and apical membrane antigen 1 (AMA1) has alsoprotected volunteers against an experimental P. falci-parum challenge [8]. Although the data from both ofthese clinical trials are not yet published, these studiesindicate that CSP, TRAP and possibly AMA1 can induceprotective immune responses in people. Unfortunately,most of the other malaria vaccine antigens evaluated inpeople have not induced significant levels of protection.For example, recombinant protein vaccines containingthe C-terminal end of the merozoite surface protein 1(MSP142), the AMA1 ectodomain or a combination ofthree P. falciparum antigens (MSP1, MSP2 and ring-infected erythrocyte surface antigen (RESA)) have notinduced significant levels of protection against naturalinfection in children living in malaria endemic regions[9-11]. Each of these vaccines, however, may haveinduced some level of strain-specific protection againstthe P. falciparum strain from which the vaccine antigenwas derived [11,12]. Since an immune response againstmultiple malaria antigens may be necessary to protect ahigh percentage of people against the large number ofantigenically diverse P. falciparum strains throughoutthe world, there is a great need to identify new malariavaccine antigens.In this report, the efficacy of three malaria vaccine

antigens was evaluated in a P. yoelii/mouse model.Although these three pre-erythrocytic stage antigens,PY03011, PY03424 and PY03661, were independentlyidentified by our bioinformatic and genomic analyses,two of the antigens (or their orthologs) were previouslydescribed (PY03011 = PyUIS3, PY03424 = falstatin)[13,14]. Protection studies with DNA and vaccinia virusvaccine vectors expressing these antigens suggest thattwo of the antigens, PY03011 and PY03424, can protectmice against a P. yoelii sporozoite challenge.

MethodsDown-selection of vaccine candidate genesP. falciparum and P. yoelii express approximately 5,800genes. It is not feasible to evaluate the vaccine potential

of that many genes. Therefore, various methods wereused to down-select the most promising vaccine candi-dates. Assuming that a vaccine based on a pre-erythro-cytic antigen is more likely to be successful than avaccine based on an erythrocytic antigen, the down-selection process focused on sporozoite and liver stageantigens. To identify promising sporozoite antigens,genomic and proteomic information contained in pre-existing malaria databases was evaluated [15,16]. Toidentify promising liver stage antigens, an expressionlibrary created with material isolated from P. yoelii-infected liver cells was evaluated [17]. The P. falciparumgenes encoding the down-selected sporozoite and liverstage antigens were cloned using a high-throughputcloning strategy [18]. Evaluation of the proteins encodedby these genes with antisera from volunteers who hadreceived a P. falciparum irradiated sporozoite vaccineidentified 20 promising vaccine candidates [19].

Generation of DNA and vaccinia virus vaccine vectorsThe re-annotated single exon PY03011 gene was isolatedfrom P. yoelii (17XNL) genomic DNA by PCR with theprimers, 5’-TGGATCCATGAAAGTGTATAAAATGAA-CACTCTC-3’ and 5’-TGGATCCTCATTTTGGTTGA-TATTGTTCTTTAAG-3’. The DNA-PY03011 vaccinevector was generated by cloning the PY03011 gene fromthis PCR reaction into the BamHI site of the DNA vaccinevector, VR1020 (Vical Inc., San Diego, CA). This cloningreaction positions the full length PY03011 gene down-stream from a cytomegalovirus (CMV) immediate-early(IE) promoter and in-frame with a human tissue plasmino-gen activator (TPA) signal sequence. Since the PY03011protein contains a signal sequence, cloning the PY03011gene into VR1020 downstream from an in-frame TPA sig-nal sequence results in a PY03011 construct that containstwo signal sequences. The vaccinia-PY03011 vaccine vec-tor was generated using a host range selection system [20].The full length PY03011 gene in this vector is insertedinto the vaccinia virus A-type inclusion body (ATI) locusand is under the transcriptional control of a syntheticearly/late (E/L) promoter [21].Exon 2 of the re-annotated PY03424 gene was isolated

from P. yoelii (17XNL) genomic DNA by PCR with theprimers, 5’-TGGATCCTACTCTTTTGACATTGTAAACGAG-3’ and 5’-TGGATCCTTATTGGACAGTTACG-TATAAAATTTTAG-3’. The DNA-PY03424 vaccinevector and the vaccinia-PY03424 vaccine vector weregenerated with the same reagents and techniques usedto generate the DNA-PY03011 and vaccinia-PY03011vectors. The DNA and vaccinia vaccine vectors expres-sing PY03424 (exon 2) do not contain the first 26codons of the PY03424 gene. Since the first 21 codonsof the PY03424 gene encode a signal sequence, thePY03424 proteins expressed by the DNA-PY03424 and

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vaccinia-PY03424 vectors do not contain the nativePY03424 signal sequence. To enhance expression, theDNA-PY03424 vector was engineered to express aPY03424 protein with a TPA signal sequence and thevaccinia-PY03424 vector was engineered to express aPY03424 protein with a human decay accelerating factor(DAF) signal sequence.The PY03661 gene was isolated from P. yoelii (17XNL)

genomic DNA by PCR with the primers, 5’-TGGATC-CATGTTTCGATCTGATTCCCATTTCC-3’ and 5’-TGGATCCTTATGTTTGATGATAATTTTCTTTCG-3’.The DNA-PY03661 vaccine vector and the vaccinia-PY03661 vaccine vector were generated with the samereagents and techniques used to generate the other DNA-P. yoelii and vaccinia-P. yoelii vectors. Since the nativePY03661 gene does not contain a signal sequence, the vac-cinia-PY03661 expression cassette was constructed with aDAF signal sequence. Therefore, the DNA-PY03661 vec-tor expresses a PY03661 protein with a TPA signalsequence and the vaccinia-PY03661 vector expresses aPY03661 protein with a DAF signal sequence.The DNA-P. yoelii vaccines were manufactured to

pre-clinical grade specifications by Puresyn, Inc. (Mal-vern, PA). The vaccinia-P. yoelii vaccines were propa-gated in RK-13 cells (rabbit kidney cells; ATCC CCL37)using standard laboratory procedures [22].

Mice and parasitesFemale CD1 outbred mice (5-6 weeks old) were pur-chased from Charles River Laboratories (Wilmington,MA). P. yoelii (17XNL non-lethal strain) parasites weremaintained by alternating passage in Anopheles stephensimosquitoes and female CD1 outbred mice.

Production of recombinant P. yoelii proteinsPY03011, PY03424 (exon 2) and PY03661 recombinantproteins were generated with a wheat germ cell-freeexpression system [23]. In brief, P. yoelii gene-specificRNA was generated from a plasmid containing an SP6-promoted P. yoelii-glutathione S-transferase (GST) taggedconstruct with SP6 RNA polymerase. The P. yoelii RNAtranscripts were translated into recombinant P. yoelii pro-tein in a wheat germ cell-free extract (CellFree SciencesCo., Yokohama, Japan). The GST-tagged P. yoelii proteinswere affinity purified with a glutathione sepharose resinand cleaved from the GST tag with tobacco etch virus pro-tease (Invitrogen Corp., Carlsbad, CA).

Generation of P. yoelii protein-specific antisera withrecombinant P. yoelii proteinsFemale CD1 mice were injected subcutaneously in thetail and scruff of the neck on days 0 and 29 with 10 μgof PY03661, PY03424 (exon 2) or PY03661 recombinantprotein adjuvanted in Montanide™ ISA720. On day 38,

the mice were bled and P. yoelii protein-specific antiseraprepared.

Indirect fluorescent antibody analysesIndirect fluorescent antibody (IFA) assays with sporo-zoite and blood stage parasites were performed as pre-viously described [24]. In brief, serial dilutions ofP. yoelii protein-specific antisera were incubated withP. yoelii (17XNL) sporozoites or blood from P. yoelii-infected mice. Parasites were visualized with a fluores-cein isothiocyanate (FITC) conjugated goat anti-mouseIgG (KPL Inc., Gaithersburg, MD). IFA analyses withliver stage parasites were performed as previouslydescribed [25]. In brief, mice were infected with P. yoeliisporozoites and livers were harvested 48 hours post-infection. P. yoelii-infected liver sections were preparedand incubated with P. yoelii protein-specific antisera.Parasites were visualized with a FITC conjugated goatanti-mouse IgG. Evans blue (0.02%) counterstain wasadded to the secondary antibody, providing a red back-ground to contrast the green FITC fluorescence whenexcited at the same wavelength.

In vitro expression analysesProtein expression from the DNA and vaccinia vectorswas evaluated by Western blot analyses. DNA-P. yoeliiplasmids were transfected into RK-13 cells with lipofec-tamine 2000CD (Invitrogen Corp., Carlsbad, CA). Vacci-nia-P. yoelii infections were performed in RK-13 cells.Cell lysates were run on 4-20% Tris-Glycine acrylamidegels (Invitrogen Corp., Carlsbad, CA), transferred toImmobilon-P polyvinylidene difluoride (PVDF) mem-branes (Millipore Corp., Bedford, MA) and probed withantisera from mice immunized with PY03011, PY03424or PY03661 vaccines. Proteins were detected with analkaline phosphatase Western-Light ChemiluminescentDetection System (Tropix Inc., Bedford, MA) and analkaline phosphatase colorimetric substrate (KPL Inc.,Gaithersburg, MD).

Protection studiesFemale CD1 mice were injected intramuscularly in thetibialis anterior muscle with 100 μl of vaccine (50 μl ineach leg) using a 0.3 ml syringe and a 29G1/2 needle(Becton Dickinson Co., Franklin Lakes, NJ) fitted with aplastic collar cut from a micropipette tip [26]. The DNAvaccine vectors were prepared in 1X Phosphate BufferedSaline (PBS) and diluted to the appropriate concentra-tion for vaccination in 1X PBS. The vaccinia vaccinevectors were prepared in 1 mM Tris (9.0) and diluted tothe appropriate concentration for vaccination in 1XPBS. Mice were challenged intravenously in the tail veinwith 300 P. yoelii (17XNL) sporozoites using a 1 ml syr-inge and 26G1/2 needle (Becton Dickinson Co., Franklin

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Lakes, NJ). Sporozoites were hand dissected frominfected mosquito salivary glands and diluted for chal-lenge in M199 medium containing 5% normal mouseserum (Gemini Bio-Products, West Sacramento, CA).In protection study 1, 14 mice per group were primed

on day 0 with 100 μg of the appropriate DNA-P. yoeliivaccine vector and boosted on day 40 with 5 × 107

plaque forming units (pfu) of the corresponding vacci-nia-P. yoelii vaccine vector. Mice immunized with acombination of vectors expressing PY03011, PY03424and PY03661 were primed with a total of 300 μg of theDNA-P. yoelii vectors and boosted with a total of 1.5 ×108 pfu of the vaccinia-P. yoelii vectors. Vaccine vectorsexpressing PyCSP were included in each study as a posi-tive control. On day 50, the mice were bled and seraprepared. On day 54, the mice were challenged with 300P. yoelii sporozoites. On days 61-68, parasitaemia wasevaluated by examining Giemsa-stained blood smears.Mice were considered positive if parasites were observedin any sample. To gauge the severity of the challenge,four groups of naïve CD1 mice were challenged withfour suboptimal doses of P. yoelii sporozoites (calculatedthrough serial dilution to be approximately 100, 33.3,11.1 or 3.7 sporozoites per mouse). From these infectiv-ity control mice, an ID50 was calculated. (An ID50, orinfectious dose 50, equals the dose of sporozoitesrequired to infect 50% of the mice.) Extrapolation fromthese results indicated that the mice injected with 300P. yoelii sporozoites were challenged with a dose equiva-lent to seven times the ID50 dose.In protection study 2, 14 mice per group were primed

on day 0 with 100 μg of the appropriate DNA-P. yoeliivaccine vector and 30 μg of a DNA vector expressingmurine granulocyte-macrophage colony-stimulating fac-tor (mGM-CSF) and boosted on day 42 with 3.3 × 107

pfu of the corresponding vaccinia-P. yoelii vaccine vec-tor. Mice immunized with two or three DNA-P. yoeliivectors were primed with a total of 200 μg or 300 μg ofthe DNA-P. yoelii vectors and 30 μg of the DNA-mGM-CSF vector and boosted with a total of 6.6 × 107 pfu or1 × 108 pfu of the vaccinia-P. yoelii vectors. Three sepa-rate groups of negative control mice were immunizedwith three different doses of DNA and vaccinia vectorsthat do not express a P. yoelii antigen. One group wasprimed with 100 μg of an “empty” DNA vector and 30μg of a DNA-mGM-CSF vector and boosted with 3.3 ×107 pfu of an “empty” vaccinia vector. A second groupwas primed with 200 μg of an “empty” DNA vector and30 μg of a DNA-mGM-CSF vector and boosted with 6.6× 107 pfu of an “empty” vaccinia vector. A third groupwas primed with 300 μg of an “empty” DNA vector and30 μg of a DNA-mGM-CSF vector and boosted with 1× 108 pfu of an “empty” vaccinia vector. On day 52, themice were bled and sera prepared. On day 57, the mice

were challenged with 300 P. yoelii sporozoites. On days64-71, parasitaemia was evaluated by examiningGiemsa-stained blood smears. Mice were consideredpositive if parasites were observed in any sample. Togauge the severity of the challenge, four groups of naivemice were challenged with four suboptimal doses ofP. yoelii sporozoites (100, 33.3, 11.1 or 3.7 sporozoites).From these infectivity control mice, it was calculatedthat the mice injected with 300 P. yoelii sporozoites inthis study were challenged with a dose equivalent to13.6 times the ID50 dose.The regimens for the two protection studies were

slightly different. For example, the dose of the individualvaccinia-P. yoelii vectors was slightly higher in protec-tion study 1 (5 × 107 pfu) than protection study 2 (3.3 ×107 pfu). Consequently, the total dose of the trivalentvaccine was 1.5 × 108 pfu in protection study 1 and 1 ×108 pfu in protection study 2. Additionally, in protectionstudy 2, the DNA vectors were mixed with a DNA-mGM-CSF plasmid. Although previous studies had indi-cated that co-administration of a DNA-PyCSP vectorwith a DNA-mGM-CSF plasmid could enhance theimmunogenicity and efficacy of a DNA-vaccinia prime-boost regimen [27], this enhancement is greater ininbred mouse strains (BALB/c and C57BL/6) thanoutbred strains [28]. Therefore, it is not surprising thatthe DNA-mGM-CSF plasmid did not appear to enhancethe efficacy of the PyCSP or trivalent P. yoelii vaccinesin protection study 2, relative to protection study 1.

Statistical analysesProtection results were analyzed by a Fisher’s Exact Testwith GraphPad Prism 5.03 software (GraphPad SoftwareInc., LaJolla, CA).

ResultsGenomic characterization of three P. yoelii vaccineantigensAnalysis of pre-erythrocytic P. falciparum proteins withsera from human volunteers immunized with a P. falci-parum irradiated sporozoite vaccine identified 20 pro-mising vaccine antigens [19]. To evaluate the vaccinepotential of these proteins in a murine protectionmodel, vaccine vectors that express the P. yoelii orthologof three of these antigens, PY03011, PY03424 andPY03661, were generated.

PY03011PY03011 is predicted by PlasmoDB [29], a Plasmodiumdatabase, to be the ortholog of the P. falciparum gene,PF13_0012. PF13_0012 is predicted by PlasmoDB to be asingle exon gene that encodes a protein that is 229 aminoacids long. PY03011 is predicted by PlasmoDB to containtwo exons and encode a protein that is 241 amino acids

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long. The first exon is predicted to encode the first 16amino acids and the second exon is predicted to encodethe remaining 225 amino acids. A previous study, how-ever, annotated the PY03011 gene to be a single exongene that encodes a protein that is 220 amino acids long[30]. The re-annotated PY03011 protein is more homolo-gous to PF13_0012 than the PlasmoDB-annotatedPY03011 protein (30% vs. 28%). The single exon annota-tion is also consistent with the annotations of the P. falci-parum, Plasmodium berghei, Plasmodium chabaudi,Plasmodium knowlesi and Plasmodium vivax orthologsof this gene, which are predicted by PlasmoDB to be sin-gle exon genes. Based upon these data, the studies in thisreport were performed with a single exon PY03011 genethat encodes a protein that is 220 amino acids long [30].The re-annotated PY03011 protein is predicted to

contain a signal sequence with a cleavage site betweenamino acids 30-31 and a transmembrane domainbetween amino acids 59-81. IFA analyses with PY03011-specific antisera indicate that PY03011 is expressed inthe sporozoite, but not in the liver or blood stages ofthe P. yoelii life-cycle (Figure 1). A previous study indi-cated that this protein was expressed in the sporozoiteand liver stages [13]. Therefore, it is likely that PY03011is expressed in the liver, but at levels that are below thelevel of detection with the serological reagents used inthe present study. The genetic characteristics of the re-annotated PY03011 gene are summarized in Table 1.

PY03424PY03424 is predicted by PlasmoDB to be the ortholog ofthe P. falciparum gene, PFI0580c. PFI0580c is predictedby PlasmoDB to contain two exons and encode a proteinthat is 413 amino acids long. The first exon is predicted toencode the first 22 amino acids and the second exon ispredicted to encode the remaining 391 amino acids.PY03424 is predicted by PlasmoDB to contain two exonsand encode a protein that is 1,856 amino acids long. Thefirst exon is predicted to encode the first 1,521 aminoacids and the second exon is predicted to encode theremaining 335 amino acids. We believe that PY03424 isnot annotated correctly and have re-annotated this gene.The re-annotated PY03424 gene is predicted to containtwo exons and encode a protein that is 357 amino acidslong. The first exon of the re-annotated gene encodes 22amino acids and the second exon encodes the remaining335 amino acids. The re-annotated PY03424 protein is sig-nificantly more homologous to PFI0580c than the Plas-moDB-annotated PY03424 protein (33% vs. 6%). Acomparison of PY03424 with other Plasmodium orthologsalso suggests that the PlasmoDB annotation is not correct.The re-annotated PY03424 protein is predicted to

contain a signal sequence with a cleavage site betweenamino acids 21-22. IFA analyses with PY03424-specific

antisera indicate that PY03424 is expressed in sporo-zoites, on the parasitophorous vacuole of the liver stageand in the blood stage (Figure 1). This profile is similarto the expression profile of the P. falciparum PFI0580cortholog, which is expressed in the sporozoite, liver andblood stages of the P. falciparum life-cycle [14,15,19].The genetic characteristics of the re-annotated PY03424gene are summarized in Table 1.

PY03661PY03661 is predicted by PlasmoDB to be the ortholog ofthe P. falciparum gene, PFC0555c. PFC0555c is predictedby PlasmoDB to be a single exon gene and encode a pro-tein that is 233 amino acids long. PY03661 is predicted tobe a single exon gene and encode a protein that is 225amino acids long. The homology between the PFC0555cand PY03661 proteins is 60%. PY03661 does not appear tocontain a signal sequence or a transmembrane domain.IFA analyses with PY03661-specific antisera indicate thatPY03661 is expressed in sporozoites, but not in the liveror blood stages (Figure 1). The P. falciparum PFC0555cortholog is expressed in the sporozoite and liver stages ofthe P. falciparum life-cycle [19]. Since PFC0555c isexpressed in the liver stage, it is likely that PY03661 is alsoexpressed in the liver, but at levels that are below the levelof detection with the serological reagents used in thisstudy. The genetic characteristics of PY03661 are summar-ized in Table 1.

Construction and analyses of DNA and vaccinia virusvaccine vectors expressing PY03011, PY03424 or PY03661DNA and vaccinia virus vaccine vectors expressing there-annotated PY03011 gene, the second exon of the re-annotated PY03424 gene or the PY03661 gene weregenerated. Western blot analyses with antigen-specificantisera indicate that the DNA (Figure 2) and vacciniavirus (Figure 3) vectors express the appropriate P. yoeliiprotein.

Protection studies in P. yoelii/mouse modelTo evaluate the vaccine potential of the three P. yoeliiantigens, CD1 outbred mice were immunized in a hetero-logous prime-boost regimen with DNA and vacciniavirus vectors that express PY03011, PY03424 orPY03661, or a combination of these vectors (Table 2). Asa positive control, mice were immunized with DNA andvaccinia vectors that express P. yoelii CSP (PyCSP). As anegative control, mice were immunized with “empty”DNA and vaccinia vectors that do not express a P. yoeliiprotein. Two weeks after the vaccinia vector boost, themice were challenged with 300 P. yoelii sporozoites.Seven through fourteen days after the challenge, protec-tion against blood stage parasitaemia was evaluated byexamining Giemsa-stained blood smears. None of the 14

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mice immunized with vectors that express PY03011 orPY03661 were sterilely protected (0% protection) andonly two of 14 mice immunized with vectors that expressPY03424 were sterilely protected (14% protection). How-ever, eight of 14 mice immunized with all three antigenswere sterilely protected (57% protection) (Figure 4).The protection elicited by these three P. yoelii antigenswas greater than the protection elicited by PyCSP (57%vs. 36%).To confirm these results and determine which combi-

nation of antigens was responsible for protection, a sec-ond efficacy study was performed. CD1 outbred micewere immunized with DNA and vaccinia virus vectorsthat express PY03011, PY03424 or PY03661 (Table 2).In this study, however, separate groups of mice wereimmunized with a combination of vectors that expressPY03011 and PY03424, or PY03424 and PY03661, or all

PY03424

PY03011

PY03661

Sporozoite Blood stageLiver stage

Figure 1 Stage-specific expression of PY03011, PY03424 and PY03661. P. yoelii sporozoite, liver stage and blood stage IFA slides wereanalyzed with antigen-specific antisera from mice immunized with PY03011, PY03424 or PY03661 recombinant proteins. Liver stage sectionswere prepared from tissue harvested 48 hours after infection with P. yoelii sporozoites. Results are not shown if expression was not detected.

Table 1 Genetic characteristics of 3 P. yoelii vaccineantigens

Gene Size Exons Signal/TM

Homology(Py vs. Pf)

Expression(Stage)

PY03011 220 a.a. 1 Yes/Yes 30% S/(L)

PY03424 357 a.a 2 Yes/No 33% S/L/B

PY03661 225 a.a. 1 No/No 60% S/(L)

The genetic structure, homology and stage-specific expression of the re-annotated PY03011 gene, the re-annotated PY03424 gene and the PlasmoDB-annotated PY03661 gene are listed. Homology (Py vs. Pf) represents theamino acid homology between the P. yoelii and P. falciparum orthologs.Expression (stage) represents the stage-specific expression of the P. yoeliiproteins. Previous studies indicated that PY03011/PyUIS3 and the P. falciparumPY03661 ortholog, PFC0555c, are expressed in the liver [13,19]. Although wedid not detect expression of PY03011 and PY03661 in the liver, it is likely thatboth proteins are expressed in the liver at levels that are below the level ofdetection with the serological reagents used in this study. To indicate thispossibility, liver stage expression of PY03011 and PY03661 is presented inparentheses. Abbreviations: a.a. = amino acids, signal = signal sequence, TM =transmembrane region, Py = P. yoelii, Pf = P. falciparum, S = sporozoite, L =liver stage, B = blood stage.

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three P. yoelii antigens. The PY03011 and PY03661 com-bination was not tested since previous studies had sug-gested that PY03424 was the primary protective antigen.As a positive control, mice were immunized with DNAand vaccinia vectors that express PyCSP. Since the miceimmunized with multiple vectors received two or threetimes more vaccine than the mice immunized with a sin-gle vector, three separate groups of negative control micewere immunized with the same amount of “empty” DNA

and vaccinia vectors as the mice that received either one,two or three vaccine vectors. Two weeks after the vacci-nia vector boost, the mice were challenged with 300 P.yoelii sporozoites. Seven through fourteen days after thechallenge, protection against blood stage parasitaemiawas evaluated by examining Giemsa-stained bloodsmears. None of the mice immunized with vectors thatexpress PY03661 were protected (0% protection) andonly one of 14 mice immunized with vectors that express

A B C D E A B C D E A B C D E

PY03011 antisera PY03424 antisera PY03661 antisera

20 -

30 -

40 -50 -60 -80 -

Figure 2 Expression of P. yoelii proteins from DNA-P. yoelii vectors. RK-13 cells were transfected with an “empty” DNA vaccine vector (laneA) or DNA vaccine vectors that express PY03011 (lane B), PY03424 (lane C), PY03661 (lane D) or PyCSP (lane E). Lysates were run on anacrylamide gel, transferred to a PVDF membrane and probed with antisera from mice immunized with DNA and vaccinia vectors that expressPY03011, PY03424 or PY03661. Arrows indicate the major P. yoelii protein products. Molecular weight markers (with kilodaltons designations) areshown in the first lane.

E

PY03424 antiseraPY03011 antisera PY03661 antisera

A B C D E A B C D E

20 -

30 -

40 -50 -60 -80 -

A B DC

Figure 3 Expression of P. yoelii proteins from vaccinia-P. yoelii vectors. RK-13 cells were infected with vaccinia vectors that express PY03011(lane A), PY03424 (lane B), PY03661 (lane C) or PyCSP (lane D), or an “empty” vaccinia vector (lane E). Lysates were run on an acrylamide gel,transferred to a PVDF membrane and probed with antisera from mice immunized with PY03011, PY03424 or PY03661 recombinant proteins.Arrows indicate the major P. yoelii protein products. Molecular weight markers (with kilodaltons designations) are shown in the first lane.

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PY03011 or PY03424 were protected (7% protection).However, six of 14 mice immunized with PY03011 andPY03424 were protected (43% protection), three of 14mice immunized with PY03424 and PY03661 were pro-tected (21% protection) and six of 14 mice immunizedwith all three P. yoelii antigens were protected (43% pro-tection) (Figure 5). The protection elicited by PY03011and PY03424 is statistically significant (PY03011/PY03424 (dose = 2X) vs. Neg con (dose = 2X), p =0.0159). Similar to the previous study, the protection eli-cited by the combination of PY03011 and PY03424 or allthree antigens was greater than the protection elicited byPyCSP (43% vs. 14%).

DiscussionIn this report, the efficacy of three pre-erythrocytic stagemalaria antigens, PY03011, PY03424 and PY03661, wasevaluated. DNA and vaccinia virus vectors expressingthese three antigens were evaluated in two P. yoelii pro-tection studies. In the first study, a trivalent vaccine thatexpressed all three antigens protected a significantlyhigher percentage of mice than vaccines that expressedeither antigen alone. Since the percentage of mice pro-tected by the trivalent vaccine (57%) exceeded the sumof the percentages protected by the univalent vaccines(14%), these results suggest a potential synergistic inter-action. In the second study, a bivalent vaccine that

Table 2 Regimens for protection studies

Protection study 1:

Prime ¬ (6 wk) ® Boost ¬ (2 wk) ® Challenge ¬ (1 wk) ® Monitor parasitaemia

Day 0 Day 40 Day 54 Days 61-68

DNA vectors (100 ug/vector) Vaccinia vectors (5 × 107 pfu/vector) 300 Py spz Blood smears

Protection study 2:

Prime ¬ (6 wk) ® Boost ¬ (2 wk) ® Challenge ¬ (1 wk) ® Monitor parasitaemia

Day 0 Day 42 Day 57 Days 64-71

DNA vectors (100 ug/vector) Vaccinia vectors (3.3 × 107 pfu/vector) 300 Py spz Blood smears

DNA-mGM-CSF (30 ug/vector)

0

10

20

30

40

50

60

70

80

% p

rote

ctio

n

100

PY03011 (dose=1X)

PY03424 (dose=1X)

PY03661 (dose=1X)

PY03011 PY03424 PY03661 (dose=3X)

PyCSP (dose=1X)

Neg con (dose=1X)

0%

14%

0%

57%

36%

0%

Figure 4 Protection study 1. Fourteen CD1 outbred mice pergroup were immunized in a prime-boost regimen with DNA andvaccinia vectors that express PY03011, PY03424 or PY03661, or acombination of vectors that express all three P. yoelii antigens.Positive control mice were immunized with DNA and vacciniavectors that express PyCSP. Negative control mice were immunizedwith DNA and vaccinia vectors that do not express a P. yoeliiantigen. Groups are designated (dose = 1X) or (dose = 3X) torepresent the relative quantity of the DNA and vaccinia vectors thatthey received. The mice were challenged with 300 P. yoeliisporozoites and evaluated for parasitaemia by examining Giemsa-stained blood smears.

0

10

20

30

40

50

60

70

80

% p

rote

ctio

n

100

PY03011 (dose=1X)

PY03424 (dose=1X)

PY03661 (dose=1X)

PY03011 PY03424 (dose=2X)

PY03424 PY03661 (dose=2X)

PY03011 PY03424 PY03661 (dose=3X)

PyCSP (dose=1X)

Neg con (dose=3X)

Neg con (dose=2X)

7% 7%

0% 0% 0%

7%14%

21%

43% 43%

Neg con (dose=1X)

Figure 5 Protection study 2. Fourteen CD1 outbred mice pergroup were immunized in a prime-boost regimen with DNA andvaccinia vectors that express PY03011, PY03424 or PY03661, or acombination of vectors that express two or all three P. yoeliiantigens. Positive control mice were immunized with DNA andvaccinia vectors that express PyCSP. Three separate groups ofnegative control mice were immunized with three different dosesof DNA and vaccinia vectors that do not express P. yoelii antigens.Groups are designated (dose = 1X), (dose = 2X) or (dose = 3X) torepresent the relative quantity of the DNA and vaccinia vectors thatthey received. The mice were challenged with 300 P. yoeliisporozoites and evaluated for parasitaemia by examining Giemsa-stained blood smears.

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expressed PY03011 and PY03424 protected an equiva-lent percentage of mice as the trivalent vaccine, suggest-ing that PY03011 and PY03424 are the primary antigensresponsible for protection. A bivalent vaccine thatexpressed PY03424 and PY03661 also protected a higherpercentage of mice than either vaccine alone. However,the level of protection induced by this bivalent vaccinewas not statistically significant relative to the PY03424,PY03661 or negative control groups. Similar to the firststudy, the number of mice protected by the trivalentvaccine (43%) or the PY03011 and PY03424 bivalentvaccine (43%) was larger than the sum protected by theunivalent vaccines (14%), a result consistent with syner-gistic protection. These studies indicate that PY03011and PY03424, and their P. falciparum orthologs, arepotential malaria vaccine antigens.PY03011 and PY03424, and/or their P. falciparum or

P. berghei orthologs, have been previously characterized.PY03011, and its P. berghei ortholog, were initially identi-fied by differential stage-specific expression studies, whichresulted in it being designated PyUIS3 (upregulated ininfectious sporozoites 3) [31,32]. Further studies indicatedthat PyUIS3/PY03011 is expressed in the liver stage parasi-tophorous vacuole, where it binds to the host cell fattyacid carrier, liver-fatty acid binding protein (L-FABP), andfacilitates the importation of fatty acids from the hepato-cyte to the parasite [13]. Down-regulation of L-FABP inhi-bits parasite growth. Therefore, although the parasite cansynthesize fatty acids, it appears that it supplements thisendogenous fatty acid production by importing fatty acidsfrom the host [13]. PyUIS3/PY03011 is homologous with afamily of Plasmodium proteins called early transcribedmembrane proteins (ETRAMPs). All of the ETRAMPsshare a similar structure; an N-terminal signal sequencefollowed by a short lysine-rich region, a second transmem-brane domain and a C-terminal region of variable length[33]. Like PyUIS3/PY03011, ETRAMPs have been shownto localize to the parasitophorous vacuole. Unlike PyUIS3/PY03011, which is expressed in the sporozoite and liverstages, many of the ETRAMPs are expressed exclusively inthe ring stage. ETRAMPs appear to localize to the liverstage or blood stage parasitophorous vacuole and havebeen shown to interact with host proteins. For example,PyUIS3/PY03011 interacts with L-FABP [13] andPFE1570w, another ETRAMP, interacts with human apoli-poproteins [34]. Therefore, this family of proteins mayinteract with multiple host proteins.A PyUIS3/PY03011-knockout parasite, Pyuis3(-), can

develop into sporozoites and invade hepatocytes, but cannot develop into merozoites, indicating that PyUIS3/PY03011 is not required for sporozoite development orsporozoite invasion of salivary glands or hepatocytes, butis required for liver stage development [35]. Mice immu-nized with Pyuis3(-) knockout parasites are protected

against a wild-type P. yoelii challenge [35]. Therefore, animmune response against PyUIS3/PY03011 is not essen-tial for protection. However, since PyUIS3/PY03011 isessential for hepatocyte development, it is not surprisingthat an immune response against this protein can helpprotect mice against a P. yoelii challenge.PFI0580c, the P. falciparum ortholog of PY03424,

encodes a putative cysteine protease inhibitor, falstatin[14]. This protein can inhibit the P. falciparum cysteineproteases, falcipain-2 and falcipain-3, as well as otherPlasmodium and human cysteine proteases. Western andmass spectrophotometry analyses indicate that PFI0580cis expressed in sporozoites, as well as the ring, schizontand merozoite stages of the P. falciparum life-cycle, butnot in trophozoites, the stage at which cysteine proteaseactivity is greatest [14]. Antibodies against falstatin caninhibit merozoite infection of erythrocytes [14]. There-fore, this protein appears to be involved in erythrocyteinvasion.The P. berghei ortholog, P. berghei inhibitor of cysteine

proteases (PbICP), has also been well characterized [36].Similar to PY03424 and PFI0580c, PbICP is expressed inmultiple stages of the parasite life-cycle. In sporozoites, itlocalizes to micronemes and is secreted by gliding sporo-zoites. In infected liver cells, it localizes to the parasito-phorous vacuole. PbICP appears to play an importantrole in both of these stages. Pre-incubation of sporozoiteswith PbICP-specific antibody inhibits sporozoite infectionof HepG2 cells. Therefore, this protein appears to play arole in sporozoite invasion of hepatocytes. In addition,HepG2 cells transfected with a plasmid expressing PbICPare resistant to apoptosis-inducing reagents. Therefore,PbICP may inhibit the programmed cell death of para-site-infected liver cells, perhaps by inhibiting one ormore of the cellular proteases involved in this process.These studies indicate that the PY03424 orthologs, falsta-tin and PbICP, play a critical role in multiple stages ofthe parasite life-cycle, including sporozoite invasion ofhepatocytes, liver stage development and merozoiteinfection of erythrocytes.It is not known what the correlates of protection are in

these studies. PyUIS3/PY03011 is expressed in the sporo-zoite and liver stages [13]. Since PyUIS3-knockout para-sites can infect hepatocytes, this protein is not requiredfor sporozoite infection of hepatocytes [35]. Therefore,antibodies against PY03011/PyUIS3 may not have animpact on sporozoite infectivity. Since PyUIS3-knockoutparasites cannot develop into functional merozoites, thisprotein is essential for liver stage development [35].PyUIS3 localizes to the liver stage parasitophorousvacuole and should not be accessible to circulating anti-bodies. Therefore, the protection induced by a PY03011-based vaccine may be more dependent on a cellularresponse than a humoral response.

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The PY03424 orthologs, falstatin and PbICP, play criti-cal roles in multiple stages of the parasite life-cycle,including sporozoite infection of hepatocytes, liver stagedevelopment and merozoite infection of erythrocytes.Antibodies against these proteins can inhibit sporozoiteinfection of hepatocytes and merozoite infection of ery-throcytes [14,36]. Therefore, PY03424-specific antibodiesmay have played a critical role in the protection observedin this study. However, since this protein also appears tobe involved in inhibiting apoptosis of infected hepato-cytes, PY03424-specific T cell responses may have alsoplayed a role in protection.The protection studies reported here were performed in

CD1 outbred mice. Although studies with other malariaantigens have indicated that higher levels of protection canbe attained in inbred mouse strains, protection is oftenantigen and strain-specific. For example, a DNA-PyCSPvaccine vector can protect BALB/c (H-2d) mice against aP. yoelii challenge, but cannot protect a high percentage ofA/J (H-2a) or B10.BR (H-2k) mice. Conversely, a DNA-PyHEP17 vaccine vector can protect A/J and B10.BR mice,but cannot protect a high percentage of BALB/c mice [37].To avoid the possibility of missing potentially protectivevaccine antigens due to HLA-restricted responses, protec-tion studies were performed in CD1 outbred mice.These results suggest that combining vaccine antigens

can have a synergistic impact on protection. Specifically,vaccine combinations with vectors that express PY03011and PY03424, or PY03011, PY03424 and PY03661 pro-tected mice at significantly higher levels than vaccinesthat express the individual antigens. Other studies havealso shown that combining vaccines can enhance pro-tection, as well as circumvent the HLA-restricted pro-tection observed with some single antigen vaccines. Forexample, a combination vaccine containing two DNAvectors that express PyCSP and PyHEP17 protected ahigher percentage of BALB/c, A/J and B10.BR mice thaneither the DNA-PyCSP or DNA-PyHEP17 vector alone[37]. In addition, monkeys immunized with DNA andvaccinia vectors expressing four P. knowlesi antigens(PkCSP, PkTRAP, PkAMA1 and PkMSP142) controlled aP. knowlesi challenge significantly better than monkeysimmunized with DNA and vaccinia vectors expressingonly PkCSP [38]. Combining vaccines, however, can haveseveral disadvantages. A multi-component vaccine maybe more expensive to manufacture than a vaccine thatcontains a single component. In addition, there is a riskthat one vaccine component can have an immunosup-pressive effect on the other components. For example, avaccine containing nine different DNA-P. falciparumvectors elicited significantly lower immune responsesagainst each individual antigen than a vaccine containingthe individual vectors [39]. Therefore, combining vaccineantigens will need to be evaluated empirically to see if

synergistic, additive or antagonistic responses areobserved.

ConclusionsThe results presented here suggest that characterizingthe protective potential of new malaria vaccine antigens,such as PY03011 and PY03424, may contribute to thedevelopment of an efficacious malaria vaccine that canovercome the antigenic diversity of malaria parasites. Infuture studies, these antigens will be tested in combina-tion with other protective antigens, such as PyCSP, tosee if even higher levels of protection can be achieved.

AcknowledgementsWe would like to thank Jessica Bolton, Joyce Wanga, Phuong Thao Pham,Nicole Barnes and Dianne Litilit for their excellent technical assistance. Theviews expressed in this paper are those of the authors and do notnecessarily reflect the official policy or position of the Department of theNavy, Department of Defense, nor the U.S. Government. This work wassupported by work unit number 6000.RAD1.F.A0309. The experimentsreported herein were conducted in compliance with the Animal Welfare Actand in accordance with the principles set forth in the “Guide for the Careand Use of Laboratory Animals”, Institute of Laboratory Animals Resources,National Research Council, National Academy Press, 1995. Thomas Richie is amilitary service member and Kalpana Gowda and Martha Sedegah areemployees of the U.S. Government. This work was prepared as part of theirofficial duties. Title 17 U.S.C. §105 provides that ‘Copyright protection underthis title is not available for any work of the United States Government.’ Title17 U.S.C. §101 defines U.S. Government work as work prepared by a militaryservice member or employee of the U.S. Government as part of thatperson’s official duties.

Author details1U.S. Military Malaria Vaccine Program, Naval Medical Research Center, 503Robert Grant Avenue, Silver Spring, MD, USA. 2Henry M. Jackson Foundationfor the Advancement of Military Medicine, 1401 Rockville Pike (Suite 600),Rockville, MD, USA. 3Department of Microbiology and Immunology,University of Maryland School of Medicine, Baltimore, MD, USA.

Authors’ contributionsKL conceived and designed the experiments. KL, JA, KG, EA and JSperformed the experiments. KL and NP analyzed the data. JA and MScontributed information and reagents. KL, NP and TR wrote the manuscript.All authors have read and approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 21 October 2010 Accepted: 16 March 2011Published: 16 March 2011

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doi:10.1186/1475-2875-10-65Cite this article as: Limbach et al.: Identification of two new protectivepre-erythrocytic malaria vaccine antigen candidates. Malaria Journal2011 10:65.

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AbstractBackgroundMethodsResultsConclusions

BackgroundMethodsDown-selection of vaccine candidate genesGeneration of DNA and vaccinia virus vaccine vectorsMice and parasitesProduction of recombinant P. yoelii proteinsGeneration of P. yoelii protein-specific antisera with recombinant P. yoelii proteinsIndirect fluorescent antibody analysesIn vitro expression analysesProtection studiesStatistical analyses

ResultsGenomic characterization of three P. yoelii vaccine antigensPY03011PY03424PY03661Construction and analyses of DNA and vaccinia virus vaccine vectors expressing PY03011, PY03424 or PY03661Protection studies in P. yoelii/mouse model

DiscussionConclusionsAcknowledgementsAuthor detailsAuthors' contributionsCompeting interestsReferencesAbstractBackgroundMethodsResultsConclusions

BackgroundMethodsDown-selection of vaccine candidate genesGeneration of DNA and vaccinia virus vaccine vectorsMice and parasitesProduction of recombinant P. yoelii proteinsGeneration of P. yoelii protein-specific antisera with recombinant P. yoelii proteinsIndirect fluorescent antibody analysesIn vitro expression analysesProtection studiesStatistical analyses

ResultsGenomic characterization of three P. yoelii vaccine antigensPY03011PY03424PY03661Construction and analyses of DNA and vaccinia virus vaccine vectors expressing PY03011, PY03424 or PY03661Protection studies in P. yoelii/mouse model

DiscussionConclusionsAcknowledgementsAuthor detailsAuthors' contributionsCompeting interestsReferences